hemp processing and analysis - university of vermont...processing, extraction & refining...
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Hemp Processing and Analysis:
Extraction, refining, and testing for
consumer potency and safety
Dr. Scot Waring
Essex, Vermont
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Overview
• Definition
• Phytocompounds of interest
• Extraction methods
• Analytical testing
• Future directions
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The same plant, but different…Brassica oleracea (wild mustard)
• Artificial selection (traits)
• Natural selection (resistance)
Same plant, different phenotypes.
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Cannabis sativa L.Taxonomy is complex
• Cannabis sativa
• C. indica
• C. ruderalis
Used for millennia
• Grain
• Oil
• Fiber
• Fuel
• Medicine
Hemp
• <0.3% Δ9-THC by dry weight (3 mg THC/g)
• Fact: Arbitrary designation (data collected from new fan leaves).
• Opinion: Inappropriate standard.
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Cannabis sativa L.Cannabis today!
• Seed oil & nutritional content
• Fiber production
• Biopolymers and bioplastics
• Phytoremediation
• Fuel biomass
• Phytochemicals
Plants are “chemical factories”
Genetics >
enzymes >
biochemical ratios (CBD:THC)
Genetics largely determine molecules produced.
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Schematic view of the biosynthetic pathways leading to the Cannabis secondary metabolites discussed in this review. Transport of
precursors is represented by dashed arrows, while direct catalytic reactions are depicted by bold arrows. See text for detailed
pathways. Abbreviations used: IPP, isopentenyl diphosphate; DMAPP, dimethylallyl diphosphate; GPP, geranyl diphosphate; FPP,
farnesyl diphosphate; MVA, mevalonate; MEP, methylerythritol phosphate.
Source: Andre, C. M., Hausman, J. F., & Guerriero, G. (2016). Cannabis
sativa: The Plant of the Thousand and One Molecules. Frontiers in plant
science, 7, 19. doi:10.3389/fpls.2016.00019
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Hemp Oil Extraction and Refining
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Processing, Extraction & RefiningProducing botanical oils from hemp
Goal: Separate and collect molecules of interest from plant material.
Solvent – dissolves molecules to make a solution.
Solventless – non-chemical separation (temperature, pressure)
Common solvents used for hemp oil extraction:
• Alcohols
• Oils and fats
• Carbon dioxide (supercritical)
• Hydrocarbons (e.g. butane, propane)
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When selecting and using a solvent consider…
• End goal (product & application)
• Expense
• Source & quality
• Safety & PPE
• Handling & storage
• Extraction system
Processing, Extraction & RefiningProducing botanical oils from hemp
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“Like dissolves like”
- your chemistry teacher probably
Solvents can be considered:
• polar (water)
• non-polar (fats, oils and CO2)
• both (alcohol)
Cannabinoids are non-polar molecules and
readily dissolve in lipids and alcohol.
Processing, Extraction & RefiningProducing botanical oils from hemp
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Lipid Extraction
• Easy and relatively inexpensive.
• Naturally incorporated into diet.
• Nutritional value can be added.
• Lipids promote absorption of
cannabinoids in intestinal tract.
Processing, Extraction & RefiningProducing botanical oils from hemp
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Carbon dioxide (CO2)
• Solid when cold (dry ice)
• Gas at room temp & pressure
• Liquid under pressure
• Supercritical fluid when under heat and pressure
• Penetrates feed material like a gas
• Has solvent properties of a liquid
• Closed-loop system
• Used to decaffeinate coffee and for dry cleaning
Processing, Extraction & RefiningProducing botanical oils from hemp
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Supercritical CO2 System Diagram
Processing, Extraction & RefiningProducing botanical oils from hemp
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Hydrocarbon extraction
• Not permitted in Vermont!
• Non-polar solvent
• Efficient and selective
• Solvent purity is low
• Highly volatile!
• Closed-loop systems only!
• Do not try this at home!
Processing, Extraction & RefiningProducing botanical oils from hemp
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Alcohol Extraction
• Ethanol (ETOH) is alcohol of choice.
• Aggressive solvent.
• Will pull out polar molecules too
(sugars and chlorophyll).
• QWET (quick wash ethanol)
• ETOH can be reclaimed and reused
and/or evaporated off.
Processing, Extraction & RefiningProducing botanical oils from hemp
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Processing, Extraction & RefiningProducing botanical oils from hemp
Refining
Winterization – adding ETOH to
extract, freezing, and cold filtering
solution in order to remove polar
molecules, fats, and waxes.
Fractional distillation – temperature
gradients used to separate hemp oil
into constituent molecules (distillate).
Decarboxylation – applying heat to
convert the acidic compounds in plant
material or oils into neutral compounds
(e.g. CBDa>CBD, CBGa>CBG).
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Processing, Extraction & RefiningFinal products
Crude – Oil that is first extracted.
May be winterized or not (20-60%).
Distillate – Oil that has been
separated using heating and
condensation to collect specific
groups of molecules (70-90%).
Isolate – A “pure” form of a
particular cannabinoid (~99.9%).
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Hemp Analytical Testing
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Hemp Analytical Testing
What is an appropriate model for
testing hemp?
• Other agricultural commodities.
• Goals: assessment of phytochemical
composition & abundance (potency)
and screening for contaminants
(safety).
• Distinction: combustion & inhalation.*
myclobutanil HCN, HCl…* not going there today
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Analytical Testing
Cannabinoid ratios matter.
A Cannabis sativa chemovar is NOT
considered to be hemp if it is:
• Chemotype I: THC dominant
• Chemotype II: THC/CBD co-dominant
C. sativa chemovars considered “HEMP”
• Chemotype III: CBD dominant
• Chemotype IV: CBD/CBG co-dominant
• Chemotype IV: no cannabinoids
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Analytical Testing
What do you want to know?
Hemp: Δ9-THC ≤ 0.3% by mass
Not Hemp: Δ9-THC > 0.3% by mass
How is this determined?
Potency using chromatography
• Liquid (HPLC)
• Gas (GC)
1. What is it?
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Analytical Testing
ISO Standard
• Testing Laboratories ISO 17025
• Traceability
• Documentation
• Solid record keeping
• Corrective Actions
Cannabis testing certification
The Emerald Test
Emerald Scientific
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Analytical Testing
Chromatography
“to write color”
Solution is separated
into different fractions
based on molecule size
and chemical properties
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Analytical TestingWhat will the results look like?
HPLC
High Performance Liquid Chromatography
major cannabinoids
Δ9-THC (low in plant)
CBD (low in plant)
THCa (dominant)
CBDa (dominant)
Analysis:
• Δ9-THC is effectively absent
• ‘Total THC’ is 0.05%
• CBD:THC ratio is 16:1
• Results:
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Analytical TestingWhat will the results look like?
HPLC
High Performance Liquid Chromatography
major cannabinoids
Δ9-THC (low in plant)
CBD (low in plant)
THCa (dominant)
CBDa (dominant)
Analysis:
• Δ9-THC is below 0.3%
• ‘Total THC’ = 5.73%*
• Results:
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Analytical TestingWhat will the results look like?
Gas Chromatography (GC)
neutral cannabinoids only
• Acidic cannabinoids (THCa, CBDa, CBGa)
are decarboxylated (THC, CBD, CBG)
during prep and volatilized into a gas.
• GC values roughly equal “total
theoretical” values on HPLC reports.
• GC can also detect and quantify
dominant terpenes.
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Analytical Testing
What do you want to know?
Answer:
We also now know the relative abundance of selected terpenes and
cannabinoids in the sampled material (potency).
1. What is it?
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Analytical Testing
What do you want to know?
• Pesticides, like myclobutanil, diazinon, and permethrin
GC-MS (gas chromatography + mass spectrometry)
• Heavy metals, like lead, cadmium, and chromium
ICP-MS (inductively coupled plasma mass spectrometry)
• Microbes, like Aspergillus spp. and E. coli
qPCR (quantitative polymerase chain reaction)
2. Is it safe to consume?
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Analytical TestingComparing qPCR to traditional agar plating
• Microbes culture broadly; beneficial and commensal included.
• Initial results in 48 to 72 hours after prep.
• Not all pathogenic microbes culture on agar.
• Does not indicate the presence of mycotoxic fungal DNA.
• Composition of microbes is “ballparked” and changes over days.
• What grows on plate is the result; # of CFUs counted as proxy.
• False positives.
• Amplifies targeted microbes only.
• Final results as soon as 2 hours after prep.
• Identifies “hit list” pathogens with high fidelity.
• Detects DNA of mycotoxic fungi (dead or alive).
• Pathogen quantification is in real time and precise.
• Ability to multiplex (multiple pathogens) in same tube
with high level of precision.
• No false positives.
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Analytical TestingComparing qPCR to traditional agar plating
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Analytical Testing
What do you want to know?
• Pesticides, like myclobutanil, diazinon, and permethrin
GC-MS (gas chromatography + mass spectrometry)
• Heavy metals, like lead, cadmium, and chromium
ICP-MS (inductively coupled plasma mass spectrometry)
• Microbes, like Aspergillus spp. and E. coli
qPCR (quantitative polymerase chain reaction)
• Mycotoxins, toxic fungal metabolites like aflatoxin and ergot
qPCR or ELISA (enzyme-linked immunosorbent assay)
2. Is it safe to consume?
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Analytical TestingHow qPCR works
1. DNA “unzipped”.
2. Taxon-specific primers adhere to ssDNA.
3. Free-floating nucleotides in solution.
4. Polymerase builds complementary strand.
5. Process repeats several times and amplifies
target sequences.
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Analytical TestingqPCR
Potential “hit list” of pathogenic
microbes on Cannabis:
• Aspergillus spp.
• Coliform spp.
• Enterobacteriaceae
• Escherichia coli
• Pseudomonas aeruginosa
• Staphylococcus aureus
• Salmonella spp.
• mycotoxins
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Analytical TestingSmall scale
• DNA-based tests
• colormetric results: +/- (red/yellow)
• male/female
• +/- THCa synthase
• +/- CBDa synthase
• +/- powdery mildew
• +/- Botrytis
• +/- russet mites
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Appreciation
Dr. Heather Darby
Cary Giguere
Stephanie Smith
Erica Cummings
Kelly McDowell, MFA
Tim Fair, Esq. and Andrew Subin, Esq.
Vermont Cannabis Solutions
Zac Eaton and Kevin McKernan